Identification and localization of myosin phosphatase in human platelets
Identification and localization of myosin phosphatase in human platelets
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DOI：
10.1042/bj3300225
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发表时间：
1998-02-15
影响因子：
4.1
通讯作者：
Hartshorne, DJ
中科院分区：
文献类型：
--
作者：
Murányi, A;Erdódi, F;Hartshorne, DJ
1型（PP 1）和2A型（PP 2A）磷酸酶活性测定在三个亚细胞级分的人血小板。约80%的活性在高速培养的上清液中。蛋白质印迹显示，PP 1（PP 1c）的催化亚基，包括α-和δ-亚型，存在于每个馏分中，但PP 2A的催化亚基的水平是非常低的低速丸（细胞骨架部分）。各种抗体检测到一个亚基类似于130 kDa的亚基（M130）的肌球蛋白磷酸酶（MP）的平滑肌在低速和高速的人类血小板颗粒。利用微囊藻毒素-琼脂糖凝胶分离PP 1c及其相关蛋白。从每个组分中分离出许多蛋白质，包括肌球蛋白、肌动蛋白和PP 1c。M130只与低速和高速弹丸分离。在未结合的组分中检测到激酶活性，并且来自低速和高速颗粒的组分分别磷酸化M130和肌球蛋白。用Calyculin A处理血小板可增加许多蛋白质的磷酸化水平，包括肌球蛋白重链和轻链，并引起细胞骨架蛋白与低速颗粒的结合，未检测到PP 1c和M130的分布的显著变化。这些结果表明，在人血小板中的MP是由PP 1c加上一个类似于平滑肌磷酸酶M130的亚基。
Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80 % of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including alpha- and delta-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low-and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low-and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy-and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet, No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.